Summary Stress is one of the most commonly reported triggers of smoking relapse. It increases frequency of smoking among chronic smokers and accelerates progression towards full relapse among abstinent smokers. This relapse risk is particularly high in the presence of other negative affective states, including anxiety, irritability, depression, and craving, especially in women. Our previous research has demonstrated altered hypothalamic-pituitary-adrenocortical (HPA) axis and endogenous opioid system (EOS) regulation of the stress response in smokers. We found that 1) smokers exhibit enhanced basal HPA activity, 2) they exhibit decreased cortisol responses to multiple acute stress procedures, and 3) early smoking relapse can be predicted by attenuated adrenocorticotropin (ACTH) and cortisol responses to stress. Recent results using an opioid blockade challenge demonstrate blunted opioid regulation of the HPA stress response in smokers relative to nonsmokers; and smoking appears to acutely normalize opioid regulation of the stress response. The clinical significance of altered opioid regulation of the stress response has not been tested in the clinical context of smoking cessation and relapse. Building on previous findings, we plan in this new study to take a novel approach in addiction relapse research by identifying indices of risk for relapse using opioid- HPA stress response patterns. Our hypothesis is that smokers who exhibit blunted HPA stress response to opioid blockade are more likely to relapse early in their cessation attempt. Blunted opioid regulation contributes to inefficient stress response and may exacerbate stress effects on craving and withdrawal symptoms. We will establish the link between altered endogenous opioid regulation of the HPA stress response, withdrawal symptoms, and craving during smoking cessation. We will develop a model to predict early smoking relapse using HPA responses to stress and HPA responses to endogenous opioid blockade. Finally, we will examine sex differences in the HPA response to stress, in the HPA response to opioid blockade, and in predictors of relapse. This research represents a step forward in translating established preclinical neurobiological models of addiction and stress. It is grounded in theory, builds on important preliminary results, and uses rigorous and reproducible procedures. Demonstrating the utility of an opioid challenge in predicting relapse is a novel direction in addiction relapse research that will enable indexing two important stress biological pathways, providing both a novel mechanism of long-term effects of tobacco addiction and a marker of treatment outcome and relapse probability. This will facilitate future efforts targeting those susceptible to effects of stress on their risk for relapse with new or existing behavioral and pharmacological treatments. Reducing relapse rates will reduce tobacco use and its devastating health effects.